Project Summary In many cell types, voltage-gate Ca2+ channels in the plasma membrane trigger intracellular Ca2+ release via ryanodine receptors (RyRs) in the endoplasmic reticulum. In skeletal muscle, a specialized form of this signaling occurs such that CaV1.1 (the principle subunit of the muscle Ca channel, also referred to as the 2+ DHPR) activates RyR1 (the skeletal RyR isoform) via a conformational interaction. A long-term goal of our research is to identify the minimum set of proteins needed for this conformational interaction, and to determine their sites of interaction with one another. Based on previous work and our own preliminary results we know that in addition to CaV1.1 and RyR1, two other proteins are required: a ?Stac? adaptor protein and junctophilin. Varying isoforms of all four proteins (CaVs, RyRs, Stacs and junctophilins) are also expressed in the nervous system and CaVs, RyRs and junctophilins are expressed in heart. Significantly, mutations of CaVs, RyRs and junctophilins give rise to inherited, human disorders of skeletal muscle, heart and the nervous system. Clearly understanding the pathogenesis of such disorders would benefit from a better understanding of how these proteins interact with one another, and a major focus of the proposed experiments is to achieve this understanding. Toward this end, we have developed a number of experimental tools, including a construct encoding only the cytoplasmic domain of RyR1 (?RyR1cyto? containing RyR1 residues 1-4300 but lacking the ~700 C-terminal residues that form the ion conducting pore and anchor RyR1 in the sarcoplasmic reticulum). Additionally, we have established the ability to obtain high level expression of CaV1.1 in non-muscle cells. With these and other results demonstrating feasibility, we propose to use cDNA expression in muscle and non- muscle cells, followed by patch clamping, confocal fluorescence microscopy, biochemistry and electron microscopy to pursue the following specific aims. Aim 1. To use expression in tsA201 cells to define the determinants for interactions between skeletal, cardiac and neuronal isoforms of the Stac proteins, the junctophilins, and RyRs. Aim 2. To exploit the differential methodological strengths of tsA201 and muscle cells for probing the molecular determinants of signaling between CaV1.1 and RyR1. Myotubes will be used to test the Stac isoform specificity for support of EC coupling and to test the role of the DHPR ?1 subunit. tsA201 cells will be used to determine how mutations affect CaV1.1 gating charge movements, and myotubes to probe proximities among constituents of the EC coupling apparatus. Aim 3. To determine whether CaV1.1-RyR1 functional interactions can be reconstituted in tsA201 cells, either partially with RyR1cyto or entirely with full-length RyR1.